Project description:The overall aim of the experiment is to understand the phenotype of mature mouse olfactory sensory neurons by analyzing the transcripts expressed and enriched in them as compared to the rest of the cell types in the olfactory epithelium (consisting of immature neurons, supporting cells, progenitor cells and cells in lamina propria) and brain ( with out the olfactory bulbs). Comparision with the other cell types in the olfactory epithelium should eliminate the transcripts commonly expressed in the olfactory epithelium and comparision with brain will eliminate the transcripts common to most neurons. Our gene chip data indicates that mature mouse olfactory sensory neurons express 10,000 genes. Mature OSNs specifically contained three clusters of over represented Gene ontology categories: smell, ion transport and cilia. Analysis for the functionally over represented categories among the transcripts with a positive signal in the mature OSNs yielded largely broad categories common to all cells with the exception of chromatin modelling and RNA processing categories. Biological process categories of movement, development and immune response came as under represented categories. Experiment Overall Design: To purify mature olfactory neurons we took advantage of the OMP-GFP mice. OMP(olfactory marker protein) is expressed specifically in mature olfactory and vomeronasal sensory neurons. In the OMP-GFP mice the coding region of OMP is replaced by GFP. We purified OSNs from the rest of the epithelium from these mice by using FACS. . We used the Affymetrix gene chips mouse expression set 430 (consisting of 430A and 430B chips). Our gene chip data is extensively validated by insitu hybridizations.
Project description:The overall aim of the experiment is to understand the phenotype of mature mouse olfactory sensory neurons by analyzing the transcripts expressed and enriched in them as compared to the rest of the cell types in the olfactory epithelium (consisting of immature neurons, supporting cells, progenitor cells and cells in lamina propria) and brain ( with out the olfactory bulbs). Comparision with the other cell types in the olfactory epithelium should eliminate the transcripts commonly expressed in the olfactory epithelium and comparision with brain will eliminate the transcripts common to most neurons. Our gene chip data indicates that mature mouse olfactory sensory neurons express 10,000 genes. Mature OSNs specifically contained three clusters of over represented Gene ontology categories: smell, ion transport and cilia. Analysis for the functionally over represented categories among the transcripts with a positive signal in the mature OSNs yielded largely broad categories common to all cells with the exception of chromatin modelling and RNA processing categories. Biological process categories of movement, development and immune response came as under represented categories. Keywords: cell type comparison
Project description:We use comprehensive and unsupervised transcriptome analyses to provide molecular classifications of sensory neurons in the mouse geniculate ganglion. 96 neurons were isolated on a C1 Fluodigm chip, underwent RNA-Seq, and iteratively clustered into sub-classes.
Project description:Identification of all genes expressed by mouse olfactory sensory neurons; genes expressed in mature neurons, immature neurons, or both were distinguished. Independent validation of enrichment ratio values supported by statistical assessment of error rates was used to build a database of statistical probabilities of the expression of all mRNAs detected in mature neurons, immature neurons, both types of neurons (shared), and the residual population of all other cell types. FACS was used to separate a highly GFP+++ sample enriched in immature neurons, a weakly GFP+ sample slightly enriched in mature neurons, and a GFP- sample that contained all cell types in the olfactory epithelium but depleted of immature neurons.
Project description:The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene function and cellular identity. To address this issue in an in vivo neuronal population, we assessed the patterning, stability, and function of the base within gene bodies in olfactory sensory neurons. We find that gene body 5hmC linearly correlates with transcriptional output and is stable in fully mature neurons and those lacking de novo methyltransferase activity. Overexpression of Tet3, which oxidizes methylated cytosines (5mC) to 5hmC, markedly alters gene body 5hmC levels and provides evidence that 5hmC facilitates transcription. This manipulation disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system that are necessary for proper physiology. Our results support a direct, positive and physiologically significant role for gene body 5hmC in transcriptional elongation and the maintenance of cellular identity independent of its function as an intermediate to demethylation. We assessed the role of 5hmC in mature olfactory sensory neurons by assessing 5hmC levels in 2 month old neurons, olfactory epithelia lacking Dnmt3a, and mOSNs overexpressing Tet3. To determine genome-wide levels of 5hmC, we performed DNA immunoprecipitation coupled to Illumina sequencing. To determine transcript levels, we prepared and sequenced rRNA-depleted cDNA libraries.
Project description:We report RNA sequencing of single olfactory neurons from mouse olfactory epithelium in developmental progression from progenitors to precursors to immature neurons to mature neurons. Most mature neurons expressed only one of ~ 1000 odorant receptor genes (Olfrs) at high levels, whereas many immature neurons expressed low levels of multiple Olfrs. Investigating expression of odorant receptors genes in mouse olfactory sensory neurons during development.
Project description:Expression profiling of mRNA abundance in the adult mouse olfactory epithelium during replacement of OSNs forced by the bilateral ablation of the olfactory bulbs. The experiment was done on 6 week old male C57Bl/6 mice. Olfactory epithelium tissue samples were collected on days 1, 5, and 7 after bulbectomy. The cellular processes activated by bulbectomy include apoptosis of mature olfactory sensory neurons, infiltration of macrophages and dendritic cells, stimulation of proliferation of basal cell progenitors, and differentation of new sensory neurons.